The yeast strain Pichia pastoris, a producer of humanized F(ab')(2) fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')(2) fragments of the shared pool of secreted immunoglobulins up to 75%.

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFP0) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS115/pPICZαA/rhAFP0, which produces unglycosylated rhAFP0 and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

Созданы штаммы спиртовых дрожжей, синтезирующие пептиды, подавляющие рост молочнокислых бактерий Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici и др., которые могут быть использованы при производстве этанола для борьбы с заражением молочнокислыми бактериями. Синтезированы гены, кодирующие антибактериальные пептиды педиоцин и плантарицин, с предпочтительными для Saccharomyces cerevisiae кодонами и создана система их секреторной экспрессии. Показано, что штаммы S. cerevisiae, синтезирующие и секретирующие в среду эти пептиды, подавляют рост молочнокислых бактерий. Использование спиртовых дрожжей, продуцирующих антибактериальные пептиды, увеличивало выход этанола в условиях бактериального инфицирования. Спиртовые дрожжи S. cerevisiae, секретирующие в среду антибактериальные пептиды педиоцин и плантарицин, могут быть рекомендованы для замены существующих промышленных линий дрожжей при производстве этанола.

A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

Physical loading or mechanical injury to muscles causes the product of the insulin-like growth factor 1 (IGF-1) gene to undergo alternative splicing, leading to production of a splice variant termed mechano-dependent growth factor (MGF). Previous in vitro experiments have demonstrated increases in the proliferative activity of myogenic precursors on exposure to MGF. However, the question of the physiological effects of MGF in the structures of normal and pathologically altered skeletal muscle remained open. We report here studies of the effects of administration of recombinant full-size MGF into the gastrocnemius muscle of rats during at 30-day recovery period after 16 weeks of alcohol intoxication. Alcoholized rats showed significant decreases in the intensity of proliferation of satellite cells, decreases in the numbers of myonuclei, and decreases in the phosphorylation of p90RSK. Studies of the level of phosphorylation of ribosomal S6 protein kinase p70S6K indicated that there were no significant changes. Administration of MGF increased the proliferative activity of myogenic precursors and restored the pool of myonuclei; the level of phosphorylation of p90RSK was also increased.

The peptide conformation in the context of a protein polypeptide chain is influenced by proximal amino acid residues. However, the mechanisms of this interference remain poorly understood. We studied the conformation of angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion from a precursor protein angiotensinogen and contain an identical peptide core structure. Using the example of angiotensins 1, 2 and 3, it was shown that similar amino acid sequences may have significant conformational differences in various molecules. In order to assess the conformational changes, we developed a panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and 3 and studied their cross-reactivity in indirect and competitive ELISAs. It was found that the conformations of inactive angiotensin1 and the corresponding fragment of angiotensinogen are similar; the same is true for the conformations of active angiotensins 2 and 3, whereas the conformations of homologous fragments in the active and inactive angiotensins differ significantly.

Вопрос о влиянии аминокислотного окружения определенных участков белка на их конформацию остается малоизученным. На примере ангиотензинов 1, 2 и 3 - метаболитов ангиотензиногена, мы показали, что одни и те же аминокислотные последовательности в составе разных молекул могут иметь существенные конформационные различия. С этой целью получены высокоаффинные моноклональные антитела против ангиотензинов 1, 2 и 3 и изучена их кросс-реактивность между разными ангиотензинами и ангиотензиногеном. Сделан вывод о том, что конформации неактивных молекул - ангиотензина 1 и соответствующего участка ангиотензиногена - сходны между собой, конформации активных ангиотензинов 2 и 3 также сходны между собой, тогда как конформации гомологичных участков у активных и неактивных ангиотензинов существенно отличаются.

In this review, we analyzed modern data on the biochemical adaptation of microorganisms to living under extreme conditions. Special attention is given to analysis of adaptation responses of microorganisms under exposure to increased radiation at molecular and cellular levels. Data on the practical use of extremophiles as well as extremoenzymes, biologically active compounds, biopolymers, etc., synthesized by them are systematized.

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.

Single-stranded DNA aptamers interacting with fibrils of Saccharomyces cerevisiae Sup35p were obtained by the SELEX procedure. The specificity of interactions with Sup35p was investigated for 10 out of 40 selected aptamers. Nine aptamers were found to bind to the fibrillar but not to the monomeric form of Sup35p. The dissociation constant of the aptamer-fibril complex ranged from 0.1 to 1.0 μM for different aptamers. The aptamers can be used to study the prion transformation of Sup35p.

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.

Recent progress in studies of anaerobic nitrate reduction and nitrous oxide formation in fungi has been reviewed. Current understanding of the biochemistry of nitrate and nitrite reduction to nitrous oxide and ammonium under oxygen limitation is presented, with emphasis on patterns of fungal co-denitrification, properties of the enzymes involved, and prevalence of nitrate respiration among fungal species.

The salt tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35–45°C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. Strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130–140 kD monomeric protein with specific activity of 250 µmol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70–80°C.

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.